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Chronic obstructive pulmonary disease (COPD) is a debilitating disease that as of yet has no cure and therapy is limited to symptomatic relief. A major risk factor for the development of COPD is smoking, although the disease does have some component of genetic predisposition. In excess of £500 million funding per year is required to accommodate the needs of COPD patients and approximately 27, 000 deaths per year in the UK can be attributed to COPD. COPD is comprised of three conditions-chronic bronchitis, bronchiolitis and emphysema and these will be present in the COPD patient to varying degrees. It is well accepted that COPD is a disease characterised by increases in inflammation and as such inflammatory stimuli, such as cigarette smoke and lipopolysaccharide (LPS) from bacterial cell walls, have been associated with disease development and progression. Consequently, there are associated increases in inflammatory cytokines such as TNFα in the COPD patient. Although inflammation plays a major role in the pathogenesis of COPD there are other factors to consider such as proteolytic damage caused by disturbances in the proteinase/anti-proteinase balance. This is of particular importance in emphysema where increases in neutrophil elastase concentration results in the destruction of elastin fibres which results in a decrease in lung function. Elastase is also known to contribute to the mucus hypersecretion associated with chronic bronchitis. The proteolytic actions of elastase are well characterised but there is gathering evidence to suggest that it may also be able to act as an inflammatory stimulus, thereby increasing its role in the pathogenesis of COPD. This study has utilised a human lung explant model to investigate whether elastase can initiate an inflammatory response; concentrations of the pro-inflammatory cytokine TNFα and the anti-inflammatory cytokine IL-10 in the culture supernatant have been investigated as part of this. Data from this model (n=36) has shown that elastase can significantly increase both TNFα and IL-10 compared to control. Elastase stimulation, for 24hrs, caused the release of 30.1±8.0pg TNFα/mg tissue and 3.1±0.5pg IL-10/mg tissue. This response is comparable to that produced by LPS. We have also found that elastase can induce a Th2 type response from the parenchymal explants, with increases in IL-4, IL-5 and IL-13. The inflammatory response detailed in this study appears to be unique to elastase and cannot be reproduced with other serine proteinases, such as trypsin and chymotrypsin, or a cysteine proteinase, papain. Our data has also shown that the proteolytic activity of elastase can be inhibited by an elastase-specific inhibitor, elastatinal, and by doing so attenuates the TNFα response; elastase stimulation alone produced 127.5±72.1pg/mg tissue, whereas with the inhibitor this production dropped to 40.4±9.0pg/mg tissue. As of yet, the exact mechanism by which elastase induces inflammation is unknown but we have investigated the relationship between elastase and two candidate receptors-proteinase-activated receptor (PAR)-2 and Toll-like receptor (TLR)-4. Although elastase stimulation does not appear to alter the gross amount of these receptors present in the parenchyma, we have found that those patients with mild to moderate COPD tend to have greater levels of both PAR-2 and TLR-4. We have also utilised synthetic activating peptides for PAR-2 and in comparison to elastase stimulation it is suggested that elastase may cause its inflammatory and Th2 effects via distinct pathways.

Neutralising antibodies are an essential component in the immune response to virus infection and are often effective at preventing or attenuating viral diseases. The administration of cocktails of potently neutralising monoclonal antibodies (nAb) to abrogate viral infections has been demonstrated as an effective treatment to control disease progression caused by a diverse range of viruses. The mechanism of neutralisation is mediated predominantly by nAb binding to glycosylated envelope proteins displayed on the surface of virus particles, thereby preventing binding to host cell membrane bound receptor proteins and neutralising the entry process. Although a robust nAb response is elicited following infection with a number of viruses, many human viruses have evolved mechanisms to effectively evade the humoral immune response. The emergence or re-emergence of pathogenic human viruses poses a significant threat to the global population. The Ebolavirus epidemic of 2014-2015 arose from the emergence of a known virus in a new area, which rapidly spread through a naïve population with devastating consequences, resulting in 11,323 fatalities from a total of 28,646 confirmed cases of infection. A rapid induction of potent, broadly nAbs was a possible pathway for infection control in the 17,323 survivors of the epidemic. The isolation of potent nAbs from seroconverted individuals following antigenic exposure provides the potential to develop novel therapeutics for administration within a region affected by an epidemic. To this end, the aim of this project was to develop a platform of methods to isolate human monoclonal antibodies directly from peripheral blood through the isolation of memory B-lymphocytes and subsequent stimulation to secrete antibodies using an in vitro culture system. Identification of DNA sequences encoding antibody heavy and light chain variable domains (VH and VL respectively), allowed for the production of recombinant antibody following the sub-cloning of VH and VL regions into plasmid expression vectors to provide a proof of principle of platform function. Secondly, the introduction of functional screens to the platform would provide a method to identify antibodies derived from memory B-cells with the ability to neutralise Ebolavirus pseudo-particles using in vitro entry inhibition assays.

Bone and joint erosion and fragility fractures are associated with osteoarthritis and osteoporosis, and represent a major unmet clinical problem. In health, the balance between chondrocytes, osteoblasts and osteoclasts is a dynamic process under tight regulation. In disease, regulation is uncontrolled resulting in overt skeletal dysfunction and bone loss. NF-κB (nuclear factor κ-light-chain-enhancer of activated B cells) is a master regulator of cellular function and is an essential element in the development and homeostasis of the skeletal system. As such it is a critical controller of chondrocyte, osteoblast and osteoclast differentiation and function. Bcl-3 (B-cell lymphoma 3-encoded protein) is an atypical IκB protein and via its selective interaction with p50 and p52 homodimers of NF-κB is a regulator of cellular function. As a regulator of NF-κB, the role of Bcl-3 was hypothesised to be critical in affecting skeletal health. With the aim of defining the role of Bcl-3 in the skeleton, mice deficient in Bcl-3 (Bcl-3-/-) were compared to wild-type (Wt) mice. Ex vivo phenotypic analysis of Bcl-3-/- mice and in vitro cellular differentiation assays of Bcl-3-/- chondrocytes, osteoblasts and osteoclasts were performed. In neonatal Bcl-3-/- mice, multiple phenotypes were discovered, including dwarfism and increased mineral density. Morphometric analysis of developing and adult mice showed that 20-week Bcl-3-/- mice had a profoundly divergent phenotype from Wt. Male 20-week Bcl-3-/- mice had increased bone density in the trabecular and cortical regions and increased biomechanical strength, compared to Wt, implicating increased bone formation. Female 20-week Bcl-3-/- mice did not differ from Wt in the trabecular region; however, they possessed decreased bone area in cortical bone. Bone turnover rates of 20-week Bcl-3-/- males were lower than in Wt, implicating an accelerated rate of bone resorption, following peak bone density. While no differences in chondrocyte differentiation were found, Bcl-3-/- osteoblasts were found to have accelerated osteogenesis. Complementarily, simulated overexpression with Bcl-3 mimetic peptide restricted osteoblast differentiation. An expression repertoire of marker genes and miRNAs reflected increased RUNX2 (Runt-related transcription factor 2) activity in early-differentiating Bcl-3-/- osteoblasts. Compared to Wt, Bcl-3-/- osteoblasts had an altered whole-transcriptomic profile in early osteogenesis, with multiple NF-κB-driven osteogenic gene clusters identified. Osteoblasts increased expression of RANKL (RANK ligand) and decreased OPG (osteoprotegerin) expression, involved in signalling cross-talk with osteoclasts. Bcl-3-/- osteoclasts had increased intrinsic osteoclastogenesis and resorption, while treatment with Bcl-3 peptide restricted osteoclast differentiation and function. The ex vivo phenotypic results were complementary to in vitro assays, showing increased bone turnover. The lack of Bcl-3 in both, osteoblasts and osteoclasts, increased their differentiation and activities. Thus, Bcl-3 functions as an inhibitor of NF-κB-driven early osteogenesis and osteoclastogenesis. These findings help identify Bcl-3 as a novel target for the treatment of diseases involving skeletal pathology.

To date there are no effective treatments for prion diseases, and these diseases are always fatal in both humans and animals. Additionally, the gold standard for diagnosis of these disease remains to be the analysis of biopsied brain tissue obtained post mortem. Consequently, there is a continued demand for therapeutics and ante-mortem diagnostics for prion diseases. This project addresses these demands by investigating candidate therapeutic and diagnostic ligands for prion diseases. This study investigated recombinant prion proteins (rPrPs) as inhibitors in scrapie and bovine spongiform encephalopathy (BSE) in vitro amplification by protein misfolding cyclic amplification (PMCA). Three ovine rPrPs with the polymorphisms VRQ, ARQ and ARR and hamster rPrP were tested against scrapie PMCA in dilution series to calculate IC50 values. The two most potent inhibitors, VRQ and ARQ, were then similarly tested against bovine spongiform encephalopathy (BSE) amplification. The most potent inhibitor of both disease types, the ovine rPrP VRQ, was then observed to inhibit a range of different scrapie and BSE strains at a fixed concentration. It is recommended that further investigation into rPrP inhibitors is performed. Strain characterisation of scrapie was investigated using rPrP inhibitors, following observations that the rPrP inhibitors generate a pattern of inhibition at a set concentration. Although this pattern of inhibition was repeatable in scrapie amplification by PMCA, this was limited to a single round of PMCA. Ultimately, this limited the application of this method to only amplification efficient prion strains and isolates. It is recommended that this method be investigated further in combination with the amplification of different isolates in substrates of different genotypes over multiple rounds of PMCA, as well as the analysis of glycoform ratios by western blotting. Here it was also identified that the imidazole used in the elution buffer for immobilised metal affinity chromatography (IMAC) can inhibit prion amplification in a strain dependent manner. This inhibition could be used in combination with the proposed method as a multi-faceted assay of prion strain characterisation. The use of next generation phage display (NGPD) to map the epitopes of autoantibodies in the sera of scrapie infected sheep was also investigated. This was performed to identify peptides that were immunoreactive to autoantibodies specific to the disease state. The identification of diagnostic peptides would then enable the development of an ante-mortem serological diagnostic test for scrapie. NGPD successfully selected immunoreactive peptides, of which 39 were selected for validation by peptide enzyme-linked immunosorbent assays (ELISAs). Although none of the peptides demonstrated diagnostic specificity by peptide ELISA, an optimised ELISA methodology was developed for future use in the validation of NGPD selected peptides. Further variations in the NGPD method, as well as validation by immunoassay, can be investigated to identify diagnostic peptides immunoreactive to scrapie specific autoantibodies.

Leishmania spp. are protozoan parasites that cause a spectrum of pathologies in humans and other vertebrates, ranging symptomatically from cutaneous ulceration to visceral dissemination. In order to survive within the host, Leishmania are able to evade and modulate the host immune responses through the actions of their virulence factors; however, few putative virulence factors have been characterised during in vivo infection with Leishmania. The Leishmania major genome has revealed the presence of three peptide inhibitors of S1A family serine peptidases (ISPs), which are orthologues of a bacterial protease inhibitor, ecotin. Serine peptidases of the S1A family are absent in Leishmania; therefore, the ISPs have been proposed to inhibit the activity of host serine peptidases, such as those expressed by cells of the innate immune response. ISP2, which is expressed in the mammalian-infective metacyclic promastigote and amastigote stages, has previously been shown to inhibit neutrophil elastase (NE), a serine peptidase expressed by neutrophils, monocytes, and macrophages. This inhibition prevents the activation of a Toll-like receptor 4 (TLR4)-NE pathway during Leishmania-macrophage interaction promoting Leishmania survival and growth in macrophages in vitro. The aims of this project were to assess whether the presence or absence of ISP2 in L. major affects parasite survival in vivo, and to investigate the effects of ISP2 on immune cell dynamics in vivo, specifically with regards to cell recruitment and activation, using the C57BL/6 mouse model. Parasite burdens were performed in mice infected with L. major wild-type (WT) parasites, a cell line deficient in ISP2/3 (Δisp2/3), and a cell line re-expressing ISP2/3 (Δisp2/3:ISP2/3). L. major Δisp2/3 parasites could not be detected at the site of inoculation by 5 wk post-infection compared with WT and Δisp2/3:ISP2/3 parasites, but parasites of all three cell lines were detected in the draining lymph nodes (dLNs) throughout the course of infection. These data were corroborated using in vivo bioluminescence imaging (BLI) of luciferase-expressing (LUC2) versions of these cell lines, in which only a low bioluminescent signal was observed at the site of inoculation with the LUC2-expressing Δisp2/3 cell line over the course of infection, compared with the LUC2-expressing L. major and Δisp2/3:ISP2/3 cell lines. These results suggest that ISP2 may be important in the establishment and persistence of Leishmania infection by conferring parasite survival, particularly at the site of infection. Serine peptidases of innate immune cells, such as NE, function in the proteolytic cleavage of cytokines, chemokines, and cell receptors, to regulate immune cell recruitment and activation. Flow cytometric analysis of innate cell populations at the site of inoculation, in response to L. major WT and Δisp2/3 parasites over 5 wk of infection, was conducted. This line of investigation revealed significantly higher numbers of monocytes, monocyte-derived macrophages, and monocyte-derived dendritic cells (moDCs) at 2 wk in Δisp2/3 infection. MoDCs have crucial functions in the induction of antigen-specific T helper 1 responses, which are considered to be important for parasite clearance. MoDCs at the site of Δisp2/3 infection showed an upregulation of the DC co-stimulatory molecule CD80 compared with those from WT infection suggesting an upregulation of DC maturation. MoDCs have also been shown to be the major producers of inducible nitric oxide synthase (iNOS) during L. major infection, which catalyses the production of nitric oxide that is responsible for the killing of Leishmania. Intracellular staining of iNOS through flow cytometric techniques showed that iNOS expression in moDCs was not affected by the presence or absence of ISP2; there was, however, an increase in iNOS expression in other innate cell types, the resident macrophages and DCs, monocytes, and monocyte-derived macrophages, at the site of Δisp2/3 infection compared with those from WT and Δisp2/3:ISP2/3 infections. At the 2 wk time-point, there was also a significant increase in the concentration of IFN-γ, a cytokine that induces iNOS expression, in response to Δisp2/3 infection compared with WT and Δisp2/3:ISP2/3 infections, as determined by ELISA. Quantitative in vivo BLI of myeloperoxidase (MPO) activity of activated phagocytes was determined over a period of 7 wk, which, also, indicated differences in phagocyte activation at the site of inoculation in L. major WT and Δisp2/3 infections. Taken together, these results indicate that the immune response is more primed towards Leishmania killing in Δisp2/3 infection compared with WT infection, which suggests that ISP2 modulates these immune responses to facilitate Leishmania survival. Infections in transgenic mice deficient in NE, Ela-/-, showed similar monocyte recruitment and moDC activation responses in Δisp2/3 infection compared with WT and Δisp2/3:ISP2/3 infections, as those observed in the C57BL/6 mice. This indicates that NE may not be the major target for ISP2 in vivo, or that there may be compensations for the loss of NE by other serine peptidases in this model. Although the exact mechanism by which ISP2 modulates the recruitment and activation of the innate immune cells in vivo remains to be determined, this study has, for the first time, shown numerous differences in the innate immune responses induced following infection with either L. major WT or a mutant deficient in a putative virulence factor using in vivo techniques, such as in vivo imaging (IVIS) and flow cytometry, to compare the infections.

Campylobacter is a major cause of acute bacterial gastroenteritis worldwide, with the highest number of infections being attributed to Campylobacter jejuni. C. jejuni is a Gram negative, spiral, motile bacterium that belongs to the campylobacterales order and is related to both Helicobacter spp. and Wolinella sp. It has long been established that proton pump inhibitors (PPIs) and other benzimidazole derivatives display anti-Helicobacter activity in vitro. PPIs have in the past been shown to affect Helicobacter pylori growth, survival, motility, morphology, adhesion/invasion potential and susceptibility to conventional antibiotics. PPIs are highly effective drugs that are well tolerated, safe for prolonged daily use and are therefore in high demand. Both the PPIs omeprazole and lansoprazole featured in the top ten drugs prescribed in England in 2014. In 2014 Campylobacter was also the most commonly diagnosed gastrointestinal infection in Scotland, in England and Wales and also in Europe. It has previously been generally accepted that patients who are being treated with PPIs are more susceptible to enteric infections such as Campylobacter than people not taking PPIs. The effect of PPI exposure on H. pylori has been investigated rigorously in the past. A single previous study has hinted that PPIs may also be capable of affecting the related organism C. jejuni,but investigations have been extremely limited in comparison to those investigating the effect of PPIs on H. pylori. This study has investigated the in vitro effects of direct contact with PPIs on the biology ofC. jejuni. Exposure to the PPI pantoprazole was found to affect C. jejuni growth/survival, motility, morphology, biofilm formation, invasion potential and susceptibility to some conventional antibiotics. Microarray studies showed that the cmeA and Cj0561c genes were significantly up-regulated in response to pantoprazole exposure and a CmeABC deficient mutant was found to be significantly more susceptible to killing by pantoprazole than was the parent strain. Proteomic analysis indicated that the oxidative stress response of C. jejuni was induced following exposure to sub-lethal concentrations of pantoprazole. C. jejuni gene expression was assessed using qRT-PCR and the genes encoding for thiol peroxidase and GroEL co-chaperonin (both involved in the C. jejuni oxidative stress response) were found to be around four times higher in response to exposure to sub-lethal concentrations of pantoprazole. Experiments using the oxidative stress inhibitors thiourea (a hydroxyl radical quencher) and bipyridyl (a ferrous iron chelator) showed that killing by pantoprazole was not mediated by hydroxyl radical production.

This thesis presents quantitative studies of T cell and dendritic cell (DC) behaviour in mouse lymph nodes (LNs) in the naive state and following immunisation. These processes are of importance and interest in basic immunology, and better understanding could improve both diagnostic capacity and therapeutic manipulations, potentially helping in producing more effective vaccines or developing treatments for autoimmune diseases. The problem is also interesting conceptually as it is relevant to other fields where 3D movement of objects is tracked with a discrete scanning interval. A general immunology introduction is presented in chapter 1. In chapter 2, I apply quantitative methods to multi-photon imaging data to measure how T cells and DCs are spatially arranged in LNs. This has been previously studied to describe differences between the naive and immunised state and as an indicator of the magnitude of the immune response in LNs, but previous analyses have been generally descriptive. The quantitative analysis shows that some of the previous conclusions may have been premature. In chapter 3, I use Bayesian state-space models to test some hypotheses about the mode of T cell search for DCs. A two-state mode of movement where T cells can be classified as either interacting to a DC or freely migrating is supported over a model where T cells would home in on DCs at distance through for example the action of chemokines. In chapter 4, I study whether T cell migration is linked to the geometric structure of the fibroblast reticular network (FRC). I find support for the hypothesis that the movement is constrained to the fibroblast reticular cell (FRC) network over an alternative 'random walk with persistence time' model where cells would move randomly, with a short-term persistence driven by a hypothetical T cell intrinsic 'clock'. I also present unexpected results on the FRC network geometry. Finally, a quantitative method is presented for addressing some measurement biases inherent to multi-photon imaging. In all three chapters, novel findings are made, and the methods developed have the potential for further use to address important problems in the field. In chapter 5, I present a summary and synthesis of results from chapters 3-4 and a more speculative discussion of these results and potential future directions.

Monoclonal antibodies are a class of therapeutic that is an expanding area of the lucrative biopharmaceutical industry. These complex proteins are predominantly produced from large cultures of mammalian cells; the industry standard cell line being Chinese Hamster Ovary (CHO) cells. A number of optimisation strategies have led to antibody titres from CHO cells increasing by a hundred-fold, and it has been proposed that a further bottleneck in biosynthesis is in protein folding and assembly within the secretory pathway. To alleviate this bottleneck, a CHO-derived host cell line was generated by researchers at the pharmaceutical company UCB that stably overexpressed two critical genes: XBP1, a transcription factor capable of expanding the endoplasmic reticulum and upregulating protein chaperones; and Ero1α, an oxidase that replenishes the machinery of disulphide bond formation. This host cell line, named CHO-S XE, was confirmed to have a high yield of secreted antibody. The work presented in this thesis further characterises CHO-S XE, with the aim of using the information gained to lead the generation of novel host cell lines with more optimal characteristics than CHO-S XE. In addition to antibodies, it was found that CHO-S XE had improved production of two other secreted proteins: one with a simple tertiary structure and one complex multi-domain protein; and higher levels of a number of endogenous protein chaperones. As a more controlled system of gene expression to unravel the specific roles of XBP1 and Ero1α in the secretory properties of CHO-S XE, CHO cells with inducible overexpression of XBP1, Ero1α, or a third gene involved in the Unfolded Protein Response, GADD34, were generated. From these cell lines, it was shown that more antibody was secreted by cells with induced overexpression of XBP1; however, Ero1α and GADD34 overexpression did not improve antibody yield. Further investigation revealed that endogenous XBP1 splicing was downregulated in the presence of an abundance of the active form of XBP1. This result indicated a novel aspect of the regulation of the activity of IRE1, the stress-induced endoribonuclease responsible for XBP1 splicing. Overall, the work described in this thesis confirms that the overexpression of XBP1 has an enhancing effect on the secretory properties of CHO cells; information which could contribute to the development of host cells with a greater capacity for antibody production.

Streptococcus pneumoniae is a major human pathogen and causes a significant burden of disease in both developed and developing countries. Currently, two pneumococcal vaccines are available, a polysaccharide conjugate vaccine for children <2 years of age and an adult polysaccharide vaccine for ‘at risk’ groups such as the elderly and immunocompromised. Unfortunately, due to the vast variation and highly recombinant nature of the pneumococcus vaccine escape through serotype replacement is significantly decreasing the efficacy of pneumococcal vaccines globally. New cost-effective and protective pneumococcal vaccines are urgently required. Pneumolysin (PLY) is a 53Kd cholesterol-dependent cytolysin that is largely conserved in all strains of Streptococcus pneumoniae, making it an ideal candidate for inclusion in a broad spectrum vaccine. It has been shown that PLY is not only a protective immunogen but also has potent adjuvant properties and stimulates both IgG and IgA antibody responses to antigens genetically coupled to the toxin (Douce et al., 2010). Both systemic and mucosal responses are induced when PLY is used as an adjuvant which may prevent colonization and therefore provide non-serotype specific herd immunity to Streptococcus pneumoniae. The cytolytic activity of PLY prevents its inclusion in a human vaccine; a non-lytic deletion mutant 76PLY was created for this purpose which retains adjuvanticity, albeit slightly reduced. The aim of this study was to elucidate the mechanism(s) of PLY/Δ6PLY adjuvanticity, it will be essential to have a basic model of adjuvant activity before PLY-based vaccines can be advanced to human clinical trials. This project used a combination of high-throughput methods such as protein pulldowns and gene expression profiling to examine the abilities of PLY, 76PLY and the truncation mutants D123PLY and D4PLY to bind to and be internalized by host cells and to differentially regulate gene expression. These studies highlighted specific and direct interactions between PLY variants and the host cytoskeleton that could mediate antigen/PLY uptake; they also revealed a pattern of gene expression that is similar to those of other adjuvants and could provide the basis for a model of adjuvanticity. Finally, through the use of reporter cell lines and transgenic TLR4-/- BMDM, the relationship between PLY and TLR4 has been further defined. A novel method for preparing vehicle controls provided evidence that the ligation of TLR4 in this system is PLY-dependent and is not an artefact caused by contaminating TLR ligands such as LPS. Once this was established it was possible to further investigate the role of TLR4 in the adjuvant activity of PLY, in particular the PLYdependent production of IL-1@. Through these studies a surprising role for TLR4 in in vitro PLY-dependent cytokine production was discovered. Additionally, it was found that complement has an essential role in the PLY-dependent production of IL-1@. The role(s) of complement and IL-1@ in the adjuvant activity were further investigated using an in vivo immunization model and the biological basis for the difference in adjuvant activity of PLY and 76PLY was defined.